Replacement of insulin-producing beta cells offers hope for cure of type 1 diabetes. Many centers world-wide, including Minnesota center are evaluating the safety and efficacy of islets for diabetes in phase 3 clinical trials. Current standard of care following islet transplantation requires lifelong use of immunosuppressive drugs to prevent rejection of transplanted islets. But use of these immunosuppressive drugs is associated with serious side effects to the patient and to the graft. The reduced ability to fight infections is one of the primary reasons why islet transplant is not considered for very young patients who would benefit the most from this emerging therapy. In addition, recent reports show that currently used immunosuppressive drugs are toxic to islets themselves and interfere with long-term survival of the graft. Therefore, to improve transplantation outcome and increase access to the patients, islet transplantation requires development of safer and more targeted protocols which will minimize the need for continuous immunosuppression. In this study, we propose a novel combination of cell- and protein-based therapy for long- term islet graft survival without the continuous use of immunosuppressive drugs. Autologous regulatory T cell (Treg) therapy offers a safer and more effective alternative to conventional immunosuppression by establishing a state of operational tolerance to the graft. Therefore, we propose to harvest autologousTregs expand in vitro and re-infuse into the patient to establish a regulatory environment that prevents graft rejection. It has been suggested that Treg therapy by itself may be insufficient to completely protect the transplanted islets as the T effector cells in diabetic patients may not respond to the suppressive influence of Tregs. Our unpublished results show that blocking costimulatory molecule, OX40 greatly reduces response of islet-specific T and enhances regulatory function of Tregs in an animal model of diabetes. In this project, we HYPOTHESIZE that the novel combination of autologous Tregs, in conjunction with OX40 blockade will facilitate immunosuppression-free graft survival in preclinical diabetes model. We will demonstrate the efficacy of combination therapy in a spontaneous diabetes model and assess its ability to prolong islet graft survival without continuous immunosuppression. By reducing the serious side effects related to the use of immunosuppressive drugs, islet transplant will become a viable therapy for majority, if not all, patients with diabetes. In addition continuous physiological levels of insulin produced by islet grafts will significantly minimize associated complications and co-morbidity, thus greatly reducing healthcare burden associated with diabetes. Overall, the results from this proposed study will enable the development and translation of an immunosuppression-free protocol for clinical islet transplantation for the cure of diabetes.